Ratiometric Methods Research Articles

Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast

Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K(+) ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.

FRET-based sensor for imaging chromium(iii) in living cells

On the basis of fluorescent resonance energy transfer from 1,8-naphthalimide to rhodamine, a fluorophore dyad (FD8) containing rhodamine and a naphthalimide moiety was synthesized as a Cr3+-selective fluorescent probe for monitoring Cr3+ in living cells with ratiometric fluorescent methods.

Intrinsic fluorescence changes associated with the conformational state of silk fibroin in biomaterial matrices

Silk fibroin is emerging as an important biomaterial for tissue engineering applications. The ability to monitor non-invasively the structural conformation of silk matrices prior to and following cell seeding could provide important insights with regards to matrix remodeling and cell-matrix interactions that are critical for the functional development of silk-based engineered tissues. Thus, we examined the potential of intrinsic fluorescence as a tool for assessing the structural conformation of silk proteins. Specifically, we characterized the intrinsic fluorescence spectra of silk in solution, gel and scaffold configurations for excitation in the 250 to 335 nm range and emission from 265 to 600 nm. We have identified spectral components that are attributed to tyrosine, tryptophan and crosslinks based on their excitation-emission profiles. We have discovered significant spectral shifts in the emission profiles and relative contributions of these components among the silk solution, gel and scaffold samples that represent enhancements in the levels of crosslinking, hydrophobic and intermolecular interactions that are consistent with an increase in the levels of ss-sheet formation and stacking. This information can be easily utilized for the development of simple, non-invasive, ratiometric methods to assess and monitor the structural conformation of silk in engineered tissues.

Color tunable, ratiometric pH sensor for high and low pH values base on 9-(cycloheptatrienylidene)fluorene derivatives

Novel absorption and fluorescence pH sensors based on 9-(cycloheptatrienylidene)fluorene (9- CHF) derivatives have been prepared by well-known Suzuki coupling reaction. 2,7-Bis(4-hydroxyphenyl)-9- CHF ( 4), 2-(4-hydroxyphenyl)-7-(naphthanlen-1-yl)-9- CHF ( 6) have the abilities to respond at both low (−1.0 → 2.0) and high (8.2 → 13.1) pH values and can be utilized in absorption, fluorescence, and wavelength ratiometric methods. They might be complements of the glass pH electrodes for pH measurement. More interestingly, 4 exhibts violet emission in low pH value range, blue emission in middle pH value range, and green emission in high pH value range, which presents a color tunable pH sensor. ▪

ZAP‐70 by flow cytometry: A comparison of different antibodies, anticoagulants, and methods of analysis

The clinical course of chronic lymphocytic leukaemia (CLL) is variable. ZAP-70 expression is believed to provide prognostic information. The flow cytometric detection of ZAP-70 is difficult because it is an intracellular antigen with weak expression in CLL. Consensus has not been reached as to the best method for measurement. We analyzed 72 CLL patient samples for ZAP-70 expression and IgVH mutational status. Sensitivity and specificity of ZAP-70 expression against IgVH mutational status were assessed for two clones (2F3.2 and 1E7.2) and for four methods of analysis: percentage positivity (PP), comparing test to isotype control, ratio of geometric means of test and isotype control, and percentage and ratiometric methods comparing test and T/NK cell populations. The effects of anticoagulant, collection times, and time to analysis were also evaluated. Sensitivity and specificity were 85 and 88%, respectively, for Upstate PP; 70 and 88% for Caltag PP; 89 and 91% for Upstate ratio; 89 and 88% for Caltag ratio. Intraobserver variability was smaller when ZAP-70 expression was assessed using a ratiometric approach rather than the percentage method. By 48 h, we observed an average decrease of 13% in the Caltag ratio in the heparin preserved samples compared to an increase of 3% in those collected in EDTA. Within the first 24-h period, a greater percent variability was observed in those samples collected into EDTA compared with heparin. Our data support a rapid method for ZAP-70 measurement using commercially available fixation/permeabilization reagents, a conjugated antibody, and a ratiometric method of analysis that minimizes subjective interpretation of the results. This is a method of ZAP-70 assessment that could be included in a routine diagnostic CLL panel; however, the choice of anticoagulant and time of analysis after collection are critical factors in accurate assessment of ZAP-70 expression.

Comparison of excitation and emission ratiometric fluorescence methods for quantifying the membrane dipole potential

We are interested in developing fluorescence methods for quantifying lateral variations in the dipole potential across cell surfaces. Previous work in this laboratory showed that the ratio of fluorescence intensities of the voltage-sensitive dye di-8-ANEPPS using excitation wavelengths at 420 and 520 nm correlates well with measurements of the dipole potential. In the present work we evaluate the use of di-8-ANEPPS and an emission ratiometric method for measuring dipole potentials, as Bullen and Saggau ( Biophys. J. 65 (1999) 2272–2287) have done to follow changes in the membrane potential in the presence of an externally applied field. Emission ratiometric methods have distinct advantages over excitation methods when applied to fluorescence microscopy because only a single wavelength is needed for excitation. We found that unlike the excitation ratio, the emission ratio does not correlate with the dipole potential of vesicles made from different lipids. A difference in the behaviour of the emission ratio in saturated compared to unsaturated lipid vesicles was noted. Furthermore, the emission ratio did not respond in the same way as the excitation ratio when cholesterol, 6-ketocholestanol, 7-ketocholesterol, and phloretin were added to dimyristoylphosphatidylcholine (DMPC) vesicles. We attribute the lack of correlation between the emission ratio and the dipole potential to simultaneous changes in membrane fluidity caused by changes in membrane composition, which do not occur when the electric field is externally applied as in the work of Bullen and Saggau. Di-8-ANEPPS can, thus, only be used via an excitation ratiometric method to quantify the dipole potential.

Accurate apoptosis measurement requires quantification of loss of expression of surface antigens and cell fragmentation

The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis. Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI). Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied. Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis.

Fundamental importance of Na+-Ca2+ exchange for the pacemaking mechanism in guinea-pig sino-atrial node.

Na+-Ca2+ exchange (NCX) current has been suggested to play a role in cardiac pacemaking, particularly in association with Ca2+ release from the sarcoplasmic reticulum (SR) that occurs just before the action potential upstroke. The present experiments explore in more detail the contribution of NCX to pacemaking. Na+-Ca2+ exchange current was inhibited by rapid switch to low-Na+ solution (with Li+ replacing Na+) within the time course of a single cardiac cycle to avoid slow secondary effects. Rapid switch to low-Na+ solution caused immediate cessation of spontaneous action potentials. ZD7288 (3 microM), to block I(f) (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 microM), to block Na+ channels, had little effect, but in the presence of either of these agents, rapid switch to low-Na+ solution again caused immediate cessation of spontaneous action potentials. Spontaneous electrical activity was also stopped following loading of the cells with the Ca2+ chelators BAPTA and EGTA, and by exposure to the NCX inhibitor KB-R7943 (5 microM). When rapid switch to low-Na+ solution caused cessation of spontaneous activity, this was found (using confocal microscopy, with fluo-4 as the Ca2+ probe) to be accompanied by an initial fall in cytosolic [Ca2+], with subsequent appearance of Ca2+ waves. Inhibition of SR Ca2+ uptake with cyclopiazonic acid (CPA, 30 microM) slowed but did not stop spontaneous activity. Rapid switch to low-Na+ solution in the presence of CPA caused abolition of spontaneous Ca2+ transients and a progressive rise in cytosolic [Ca2+]. With ratiometric fluorescence methods (indo-5F as the Ca2+ probe), the minimum level of [Ca2+] between beats was found to be approximately 225 nM, and abolition of beating with nifedipine, acetylcholine or adenosine caused a fall in cytosolic [Ca2+] below this level. These observations support the hypothesis that NCX current is essential for normal pacemaker activity under the conditions of our experiments. A continuous depolarizing influence of current through the NCX protein might result from maintained electrogenic NCX (with 3:1 stoichiometry, supported by a cytosolic [Ca2+] that normally does not fall below 225 nM between beats) and/or from a novel, recently suggested role of the NCX protein to allow a Na+ leak pathway.

Photophysics of a Series of Efficient Fluorescent pH Probes for Dual‐Emission‐Wavelength Measurements in Aqueous Solutions

This paper evaluates the 5-aryl-2-pyridyloxazole backbone to engineer donor-acceptor fluorescent pH probes after one- or two-photon absorption. Parent fluorophores, as well as derivatives that can be used to label biomolecules, can be easily obtained in good yields. These molecules exhibit a large one-photon absorption in the near-UV range, and a strong fluorescence emission that covers the whole visible domain. The 5-aryl-2-pyridyloxazole derivatives also possess significant cross sections for two-photon absorption. Upon pyridine protonation, large shifts were observed in the absorption spectra after one- and two-photon excitation, as well as in the emission spectra. This feature was used to measure the pK(a) of the investigated compounds that range between 2 and 8. In most of the investigated derivatives, the pK(a) increased upon light excitation and protonation exchanges took place during the lifetime of the excited state, as shown by phase-modulation fluorometry analysis. Several 5-aryl-2-pyridyloxazole derivatives are suggested as efficient probes to reliably measure the pH of aqueous solutions by means of ratiometric methods that are dependent on fluorescence emission.

A wide dynamic range detection of biopolymer medicines with resonance light scattering and absorption ratiometry

Using the interaction of heparin with Janus Green Blue (JGB) as an example, herein a dual-wavelength resonance lighting scattering (RLS) ratiometric method and an absorbance ratiometric one are developed to detect biopolymer medicines based on their bindings with organic dyes. In aqueous solution, heparin could interact with JGB, displaying significantly enhanced RLS signals in UV–vis region. By measuring the RLS intensity ratio ( R ratio) and absorbance ratio ( A ratio) at the wavelengths of 285 nm over that at 345 nm, respectively, heparin over a wide dynamic range of content could be detected. Typically, when JGB concentration is kept at 1.00 × 10 −5 mol l −1, using R ratio could detect heparin over a range of 0.30–2000 ng ml −1 with a limit of determination of 30 pg ml −1, while using A ratio could detect heparin over the range of 0.01–2000.0 ng ml −1 with a limit of determination of 1 pg ml −1. The wavelength-dependent RLS and absorption ratiometric methods were found to have much better flexibility for a series of common influences, such as pH, and dye concentration, than that of RLS method at a single wavelength. The interaction nature was investigated through size measurements and RLS imaging detections.

Fluorescence studies of single biomolecules

Single-molecule fluorescence has the capability to detect properties buried in ensemble measurements and, hence, provides new insights about biological processes. Ratiometric methods are normally used to reduce the effects of excitation beam inhomogeneity. Fluorescence resonance energy transfer is widely used but there are problems in inserting the fluorophores in the correct position on the biomolecule, particularly if the structure is not known. We have recently developed two-colour coincidence single-molecule fluorescence that addresses this problem. This method can be used to determine quantitatively the multimerization states of biomolecules, in solution without separation. The future prospects of single-molecule fluorescence as applied to biological molecules are discussed.

Ln(III)-DOTAMGly complexes: a versatile series to assess the determinants of the efficacy of paramagnetic chemical exchange saturation transfer agents for magnetic resonance imaging applications.

Paramagnetic Ln-DOTAMGly complexes (Ln not equal La, Lu, and Gd) are the prototypes of a novel class of contrast agents for magnetic resonance imaging based on chemical exchange saturation transfer (CEST). Their ability to reduce the water signal intensity depends on the interplay of several physico-chemical properties of the agent and instrumental parameters. This study aims to identify possible routes for their optimization Saturation transfer (ST) has been measured in vitro at 7.05 T as a function of pH, temperature, and concentration of the agent. Large saturation transfer effects have been observed upon irradiating the coordinated water protons (for Ln = Pr, Nd, Eu, and Tb). The comparison of the results obtained by irradiating water versus amide protons allows the set-up of ratiometric methods through which the ST response can be made independent on the concentration of the agent. The modulation of the magnetic properties along the lanthanide series allows an in-depth understanding of the determinants of ST effect and provides useful insights for the design of more efficient agents.

Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring

Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells.MethodsPC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining for chemotherapeutic action of different drugs.ResultsDNA fragmentation, ratiometric ions and fluorescence endlabelling plots were characteristic for cell lines and drug response. 31P-23Na NMR spectra showed characteristic high phospho-choline and sodium peaks.ConclusionFlow cytometry, DNA fragmentation, ion ratiometric methods and NMR peaks indicated apoptosis and offered in vivo drug monitoring method.

Narrow-range optical pH sensors based on luminescent europium and terbium complexes immobilized in a sol gel glass.

The metal-based emission of a series of luminescent europium and terbium complexes incorporating an aromatic chromophore is rendered pH-dependent either through perturbation of the aryl singlet or triplet energy or by modulating the degree of quenching of the lanthanide excited state. Systems exhibiting each of these pathways have been incorporated in thin-film sol gel matrixes and evaluated as pH sensors in static and flow analyses at constant ionic strength. pH-dependent intensity or ratiometric methods, for emission or excitation spectra, have been defined for lanthanide complexes incorporating substituted phenanthridine (pK(a) from ca. 6.8 to 7.2) or 6-cyanophenanthridine-2-sulfonyl chromophores (pK(a) approximately 7.14 in human serum solution) (lambda(exc) 365 nm, phi H(2)O = 7.2%).

Imaging cytosolic free-calcium distribution and oscillations in pollen tubes with confocal microscopy: A comparison of different dyes and loading methods

A steep, oscillating tip-focused gradient in cytosolic free calcium ([Ca2+]c) has been implicated in pollen tube growth. Further understanding of the biological causes and consequences of these processes relies on the precise imaging of [Ca2+]c during the different growth phases. In this work, the minimum technical requirements for confocal [Ca2+]c imaging ofAgapanthus umbellatus pollen tubes were examined. A range of dyes, dye forms, and loading methods were compared. Non-ratio and ratio imaging were critically analysed, in terms of the detection of the [Ca2+]c gradient and its fluctuations over time. Both ratiometric and nonratiornetric methods detected relative changes in [Ca2+]c. However, visualisation of the [Ca2+]c gradient, with an accurate spatial definition, was only possible with ratiometric methods. The gradient observed in this study ranged from 1.8 μM (tip) to 180–220 nM (basal level), within the first 4–10 μm. Apical [Ca2+]c fluctuations with an amplitude between 415 nM and 1.8 μM showed a period of 40 to 75 s. All protocols for dye-loading proved to have strengths and weaknesses. Thus, the choice of a dye and its loading procedure should consider the required imaging period, extent of sequestration, effect on cell performance and viability, ease of loading procedure, and aim of the study. The present study constitutes an examination of the [Ca2+]c gradient in pollen tubes by these criteria.

Estimating Intracellular Calcium Concentrations and Buffering without Wavelength Ratioing

We describe a method for determining intracellular free calcium concentration ([Ca 2+]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca 2+] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca 2+] indicators for which saturation can be achieved under physiological conditions. In addition, the method requires that the indicators have large dynamic ranges. Popular indicators such as Calcium Green-1 or Fluo-3 fulfill these conditions. As a test of the method, we measured [Ca 2+] in CA1 pyramidal neurons in rat hippocampal slices using Oregon Green BAPTA-1 and 2-photon laser scanning microscopy (BAPTA: 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid). Resting [Ca 2+] was 32–59 nM in the proximal apical dendrite. Monitoring action potential-evoked [Ca 2+] transients as a function of indicator loading yielded estimates of endogenous buffering capacity (44–80) and peak [Ca 2+] changes at zero added buffer (178–312 nM). In young animals (postnatal days 14–17) our results were comparable to previous estimates obtained by ratiometric methods (Helmchen et al., 1996, Biophys. J. 70:1069–1081), and no significant differences were seen in older animals (P24–28). We expect our method to be widely applicable to measurements of [Ca 2+] and [Ca 2+]-dependent processes in small neuronal compartments, particularly in the many situations that do not permit wavelength ratio imaging.